Exonuclease I

• Excises bases on single-stranded DNA in 3ʹ→ 5ʹ direction
• Removes linear single-stranded DNA, leaving behind double-stranded DNA in the sample

Exonuclease I cleaves single-stranded DNA in the 3ʹ→5ʹ direction, releasing 5ʹ mono/di-nucleotides and leaving double-stranded DNA molecules and the 5ʹ terminus intact. The enzyme is processive though digestion is inhibited by a 3ʹ terminal phosphate. Exonuclease I is tolerant of a wide range of buffer conditions and can typically be added to reactions containing magnesium (1–3).

This enzyme is supplied in 10 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.5 mM EDTA and 50% glycerol: pH 7.5 at 25°C.

• Storage temperature: –25°C to –15°C
• Molecular weight: 54,500 Daltons

The protein is produced by a strain of E. coli that expresses the recombinant Exonuclease I gene.

One unit is defined as the amount of enzyme required to produce 10 nmol of acid-soluble total nucleotide in 30 minutes at 37°C.

Usage Instructions 

Removal of single-stranded DNA from PCR products 

1. Set up the following reaction mixture in a total volume of 20 µL:
   • 5 µL of PCR product 
   • 10 units of Exonuclease I (X8010L)
2. Incubate the reaction mixture at 37°C for 15 minutes. 
3. Heat inactivate enzyme at 80°C for 15 minutes.

Notes: 

1. Exonuclease I will preferentially degrade single-stranded oligonucleotide primers in a reaction containing amplification products or other sources of double-stranded DNA, leaving double-stranded molecules intact.
2. Exonuclease I, combined with Lambda Exonuclease (X8030L), effectively removes linear DNA species from plasmid preparations. 
3. Exonuclease I works well in most molecular biology buffers that contain magnesium over 1.5 mM. 

References: 

1. Lehman, I.R. and Nussbaum, A.L. (1964) J. Biol. Chem., 239, 2628. 
2. Kushner, S.R. et al. (1971) Proc. Natl. Acad. Sci. USA, 68, 824. 
3. Kushner, S.R. et al. (1972) Proc. Natl. Acad. Sci. USA, 69, 1366. 

Quality Control 

Unit activity was measured using a twofold serial dilution method. 

Dilutions of the enzyme were made in an Exonuclease I storage solution containing glycerol (50%) and added to 50 µL reactions containing a single-stranded tritiated DNA fragment and 67 mM glycine-KOH (pH 9.5), 10mM DTT, 6.7 mM MgCl₂. Reactions were incubated for 10 minutes at 37°C, placed on ice, and analyzed using a TCA-precipitation method.

Protein concentration is determined by OD₂₈₀ absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the band's mass corresponding to the protein of interest in the diluted sample.

Double-stranded endonuclease activity is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µL replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

This product is available for molecular biology applications such as:

• Plasmid prep cleanup
• Removes ssDNA from a mixture of linear ssDNA and dsDNA
• Removes oligonucleotide primers after PCR